U.S. patent application number 10/390206 was filed with the patent office on 2003-09-04 for non-nucleotide containing enzymatic nucleic acid.
This patent application is currently assigned to Ribozyme Pharmaceuticals, Inc.. Invention is credited to Beigelman, Leonid, Karpeisky, Alexander, Matulic-Adamic, Jasenka, Usman, Nassim, Wincott, Francine.
Application Number | 20030165969 10/390206 |
Document ID | / |
Family ID | 27381788 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165969 |
Kind Code |
A1 |
Usman, Nassim ; et
al. |
September 4, 2003 |
Non-nucleotide containing enzymatic nucleic acid
Abstract
Enzymatic nucleic acid molecule containing one or more
non-nucleotide mimetics, and having activity to cleave an RNA or
DNA molecule.
Inventors: |
Usman, Nassim; (Boulder,
CO) ; Wincott, Francine; (New York, NY) ;
Matulic-Adamic, Jasenka; (Boulder, CO) ; Beigelman,
Leonid; (Longmont, CO) ; Karpeisky, Alexander;
(Lafayette, CO) |
Correspondence
Address: |
Anita J. Terpstra, Ph.D.
McDonnell Boehnen Hulbert & Berghoff
32nd Floor
300 S. Wacker Drive
Chicago
IL
60606
US
|
Assignee: |
Ribozyme Pharmaceuticals,
Inc.
|
Family ID: |
27381788 |
Appl. No.: |
10/390206 |
Filed: |
March 17, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10390206 |
Mar 17, 2003 |
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10041614 |
Jan 8, 2002 |
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6555668 |
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10390206 |
Mar 17, 2003 |
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09628959 |
Jul 31, 2000 |
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6362323 |
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10390206 |
Mar 17, 2003 |
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09182975 |
Oct 29, 1998 |
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6117657 |
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10390206 |
Mar 17, 2003 |
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08363253 |
Dec 23, 1994 |
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5891683 |
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10390206 |
Mar 17, 2003 |
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08233748 |
Apr 19, 1994 |
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10390206 |
Mar 17, 2003 |
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08152481 |
Nov 12, 1993 |
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10390206 |
Mar 17, 2003 |
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08116177 |
Sep 2, 1993 |
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Current U.S.
Class: |
435/6.14 ;
536/23.1 |
Current CPC
Class: |
C12N 2310/3521 20130101;
C12N 2310/318 20130101; C07H 21/00 20130101; C12N 15/1137 20130101;
C07H 19/06 20130101; C12N 2310/322 20130101; C07H 19/20 20130101;
C12N 2310/346 20130101; C12N 15/113 20130101; C12N 2310/315
20130101; C12N 2310/332 20130101; C12Y 304/24017 20130101; C12N
2310/121 20130101; C07H 19/10 20130101; C12N 15/111 20130101; C07H
19/16 20130101; C12N 2310/33 20130101; C12N 2330/30 20130101; C12N
2310/32 20130101; C12N 15/1138 20130101; C12N 2310/321 20130101;
C12N 2310/321 20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02 |
Claims
1. A double stranded RNA molecule comprising at least one of the
non-nucleotide moieties selected from the group consisting of:
1wherein, said "n" is an integer of between 1 and 10; X is
independently oxygen, nitrogen, sulfur or substituted carbons
including alkyl or alkene; R is independently an H, alkyl, alkenyl
or alkynyl of 1-10 carbon atoms; and Y is independently a
phosphodiester, ether or amide linkage to the nucleic acid
molecule.
2. A double stranded RNA molecule comprising at least one
non-nucleotide moiety, wherein said non-nucleotide moiety lacks a
nucleic acid base and wherein said non-nucleotide comprises a
nucleotide sugar and phosphate component.
3. The double stranded RNA molecule of claim 1, wherein said RNA
comprises one or more chemical modifications.
4. The double stranded RNA molecule of claim 2, wherein said RNA
comprises one or more chemical modifications.
5. The double stranded RNA molecule of claim 1, wherein said
non-nucleotide covalently links one strand of said molecule to
another strand of said molecule.
6. The double stranded RNA molecule of claim 2, wherein said
non-nucleotide covalently links one strand of said molecule to
another strand of said molecule.
Description
[0001] This application is a continuation of Usman et al., U.S.
Ser. No. 10/041,614 filed Jan. 8, 2002, which is a continuation of
Usman et al., U.S. Ser. No. 09/628,959, filed Jul. 31, 2000, now
U.S. Pat. No. 6,362,323, which is a continuation of Usman et al.,
U.S. Ser. No. 09/182,975, filed Oct. 29, 1998, now U.S. Pat. No.
6,117,657, which is a continuation of Usman et al., U.S. Ser. No.
08/363,253, filed Dec. 23, 1994, now U.S. Pat. No. 5,891,683, which
is a continuation-in-part of Usman et al., U.S. Ser. No.
08/233,748, filed Apr. 19, 1994, now abandoned, which is a
continuation-in-part of Usman et al., U.S. Ser. No. 08/152,481,
filed Nov. 12, 1993, now abandoned, which is a continuation-in-part
of Usman et al., U.S. Ser. No. 08/116,177, filed Sep. 2, 1993, now
abandoned, all entitled "Non-Nucleotide Containing Enzymatic
Nucleic Acid" and all hereby incorporated by reference herein
(including drawings).
BACKGROUND OF THE INVENTION
[0002] This invention relates to chemically synthesized
non-nucleotide-containing enzymatic nucleic acid.
[0003] The following is a brief history of the discovery and
activity of enzymatic RNA molecules or ribozymes. This history is
not meant to be complete but is provided only for understanding of
the invention that follows. This summary is not an admission that
all of the work described below is prior art to the claimed
invention.
[0004] Prior to the 1970s it was thought that all genes were direct
linear representations of the proteins that they encoded. This
simplistic view implied that all genes were like ticker tape
messages, with each triplet of DNA "letters" representing one
protein "word" in the translation. Protein synthesis occurred by
first transcribing a gene from DNA into RNA (letter for letter) and
then translating the RNA into protein (three letters at a time). In
the mid 1970s it was discovered that some genes were not exact,
linear representations of the proteins that they encode. These
genes were found to contain interruptions in the coding sequence
which were removed from, or "spliced, out" of, the RNA before it
became translated into protein. These interruptions in the coding
sequence were given the name of intervening sequences (or introns)
and the process of removing them from the RNA was termed splicing.
At least three different mechanisms have been discovered for
removing introns from RNA. Two of these splicing mechanisms involve
the binding of multiple protein factors which then act to correctly
cut and join the RNA. A third mechanism involves cutting and
joining of the RNA by the intron itself, in what was the first
discovery of catalytic RNA molecules.
[0005] Cech and colleagues were trying to understand how RNA
splicing was accomplished in a single-celled pond organism called
Tetrahymena thermophila. Cech proved that the intervening sequence
RNA was acting as its own splicing factor to snip itself out of the
surrounding RNA. Continuing studies in the early 1980's served to
elucidate the complicated structure of the Tetrahymena intron and
to decipher the mechanism by which self-splicing occurs. Many
research groups helped to demonstrate that the specific folding of
the Tetrahymena intron is critical for bringing together the parts
of the RNA that will be cut and spliced. Even after splicing is
complete, the released intron maintains its catalytic structure. As
a consequence, the released intron is capable of carrying out
additional cleavage and splicing reactions on itself (to form
intron circles). By 1986, Cech was able to show that a shortened
form of the Tetrahymena intron could carry out a variety of cutting
and joining reactions on other pieces of RNA. The demonstration
proved that the Tetrahymena intron can act as a true enzyme: (i)
each intron molecule was able to cut many substrate molecules while
the intron molecule remained unchanged, and (ii) reactions were
specific for RNA molecules that contained a unique sequence (CUCU)
which allowed the intron to recognize and bind the RNA. Zaug and
Cech coined the term "ribozyme" to describe any ribonucleic acid
molecule that has enzyme-like properties.
[0006] Also in 1986, Cech showed that the RNA substrate sequence
recognized by the Tetrahymena ribozyme could be changed by altering
a sequence within the ribozyme itself. This property has led to the
development of a number of site-specific ribozymes that have been
individually designed to cleave at other RNA sequences.
[0007] The Tetrahymena intron is the most well-studied of what is
now recognized as a large class of introns, Group I introns. The
overall folded structure, including several sequence elements, is
conserved among the Group I introns, as is the general mechanism of
splicing. Like the Tetrahymena intron, some members of this class
are catalytic, i.e., the intron itself is capable of the
self-splicing reaction. Other Group I introns require additional
(protein) factors, presumably to help the intron fold into and/or
maintain its active structure.
[0008] Ribonuclease P (RNaseP) is an enzyme comprised of both RNA
and protein components which are responsible for converting
precursor tRNA molecules into their final form by trimming extra
RNA off one of their ends. RNaseP activity has been found in all
organisms tested. Sidney Altman and his colleagues showed that the
RNA component of RNaseP is essential for its processing activity;
however, they also showed that the protein component also was
required for processing under their experimental conditions. After
Cech's discovery of self-splicing by the Tetrahymena intron, the
requirement for both protein and RNA components in RNaseP was
reexamined. In 1983, Altman and Pace showed that the RNA was the
enzymatic component of the RNaseP complex. This demonstrated that
an RNA molecule was capable of acting as a true enzyme, processing
numerous tRNA molecules without itself undergoing any change.
[0009] The folded structure of RNaseP RNA has been determined, and
while the sequence is not strictly conserved between RNAs from
different organisms, this higher order structure is. It is thought
that the protein component of the RNaseP complex may serve to
stabilize the folded RNA in vivo.
[0010] Symons and colleagues identified two examples of a
self-cleaving RNA that differed from other forms of catalytic RNA
already reported. Symons was studying the propagation of the
avocado sunblotch viroid (ASV), an RNA virus that infects avocado
plants. Symons demonstrated that as little as 55 nucleotides of the
ASV RNA was capable of folding in such a way as to cut itself into
two pieces. It is thought that iii vivo self-cleavage of these RNAs
is responsible for cutting the RNA into single genome-length pieces
during viral propagation. Symons discovered that variations on the
minimal catalytic sequence from ASV could be found in a number of
other plant pathogenic RNAs as well. Comparison of these sequences'
revealed a common structural design consisting of three stems and
loops connected by a central loop containing many conserved
(invariant from one RNA to the next) nucleotides. The predicted
secondary structure for this catalytic RNA reminded the researchers
of the head of a hammer; thus it was named as such.
[0011] Uhlenbeck was successful in separating the catalytic region
of the ribozyme from that of the substrate. Thus, it became
possible to assemble a hammerhead ribozyme from 2 (or 3) small
synthetic RNAs. A 19-nucleotide catalytic region and a
24-nucleotide substrate were sufficient to support specific
cleavage. The catalytic domain of numerous hammerhead ribozymes
have now been studied by both the Uhlenbeck's and Symons' groups
with regard to defining the nucleotides required for specific
assembly and catalytic activity, and determining the rates of
cleavage under various conditions.
[0012] Haseloff and Gedach showed it was possible to divide the
domains of the hammerhead ribozyme in a different manner. By doing
so, they placed most of the required sequences in the strand that
did not get cut (the ribozyme) and only a required UH where H=C, A,
or U in the strand that did get cut (the substrate). This resulted
in a catalytic ribozyme that could be designed to cleave any UH RNA
sequence embedded within a longer "substrate recognition" sequence.
The specific cleavage of a long mRNA, in a predictable manner using
several such hammerhead ribozymes, was reported in 1988.
[0013] One plant pathogen RNA (from the negative strand of the
tobacco ringspot virus) undergoes self-cleavage but cannot be
folded into the consensus hammerhead structure described above.
Bruening and colleagues have independently identified a
50-nucleotide catalytic domain for this RNA. In 1990, Hampei and
Tritz succeeded in dividing the catalytic domain into two parts
that could act as substrate and ribozyme in a multiple-turnover,
cutting reaction. As with the hammerhead ribozyme, the catalytic
portion contains most of the sequences required for catalytic
activity, while only a short sequence (GUC in this case) is
required in the target. Hampel and Tritz described the folded
structure of this RNA as consisting of a single hairpin and coined
the term "hairpin" Ribozyme (Bruening and colleagues use the term
"paperclip" for this ribozyme motif). Continuing experiments
suggest an increasing number of similarities between the hairpin
and hammerhead ribozymes in respect to both binding of target RNA
and mechanism of cleavage.
[0014] Hepatitis Delta Virus (HDV) is a virus whose genome consists
of single-stranded RNA. A small region (about 80 nucleotides) in
both the genomic RNA, and in the complementary anti-genomic RNA, is
sufficient to support self-cleavage. In 1991, Been and Perrotta
proposed a secondary structure for the HDV RNAs that is conserved
between the genomic and anti-genomic RNAs and is necessary for
catalytic activity. Separation of the HDV RNA into "ribozyme" and
"substrate" portions has recently been achieved by Been. Been has
also succeeded in reducing the size of the HDV ribozyme to about 60
nucleotides.
[0015] Table I lists some of the characteristics of the ribozymes
discussed above.
[0016] Eckstein et al., Intemati0nal Publication No. WO 92/07065;
Perrault et al., Nature 1990, 344:565; Pieken et al., Science 1991,
253:314; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17:334;
and Rossi et al., International Publication No. WO 91/03162,
describe various chemical modifications that can be made to the
sugar moieties of enzymatic nucleic acid molecules.
[0017] Usman, et al., WO 93/15187 in discussing modified structures
in ribozymes states:
[0018] It should be understood that the linkages between the
building units of the polymeric chain may be linkages capable of
bridging the units together for either in vitro or in vivo. For
example the linkage may be a phosphorous containing linkage, e.g.,
phosphodiester or phosphothioate, or may be a nitrogen containing
linkage, e.g., amide. It should further be understood that the
chimeric polymer may contain non-nucleotide spacer molecules along
with its other nucleotide or analogue units. Examples of spacer
molecules which may be used are described in Nielsen et al.
Science, 254:1497-1500 (1991).
[0019] Jennings et al., WO 94/13688 while discussing hammerhead
ribozymes lacking the usual stem II base-paired region state:
[0020] One or more ribonucleotides and/or deoxydbonucleotides of
the group (X)m, [stem II] may be replaced, for example, with a
linker selected from optionally substituted polyphosphodiester
(such as poly(1-phospho-3-propanol)), optionally substituted alkyl,
optionally substituted polyamide, optionally substituted glycol,
and the like. Optional substituents are well known in the art, and
include alkoxy (such as methoxy, ethoxy and propoxy), straight or
branch chain lower alkyl such as C1-C5 alkyl), amine, aminoalkyl
(such as amino C1-C5 alkyl), halogen (such as F, Cl and Br) and the
like. The nature of optional substituents is not of importance, as
long as the resultant endonuclease is capable of substrate
cleavage.
[0021] Additionally, suitable linkers may comprise polycyclic
molecules, such as those containing phenyl or cyclohexyl rings. The
linker (L) may be a polyether such as polyphosphopropanediol,
polyethyleneglycol, a bifunctional polycyclic molecule such as a
bifunctional pentalene, indene, naphthalene, azulene, heptalene,
biphenylene, asymindacene, sym-indacene, acenaphthylene, fluorene,
phenalene, phenanthrene, anthracene, fluoranthene,
acephenathrylene, aceanthrylene, triphenylene, pyrene, chrysene,
naphthacene, thianthrene, isobenzofuran, chromene, xanthene,
phenoxathiin, indolizine, isoindole, 3-H-indole, indole,
1-H-indazole, 4-H-quinolizine, isoquinoline, quinoline,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, 4-.alpha.H-carbzole, carbazole, B-carboline,
phenanthridine, acridine, perimidine, phenanthroline, phenazine,
phenolthiazine, phenoxazine, which polycyctic compound may be
substituted or modified, or a combination of the polyethers and the
polycyclic molecules.
[0022] The polycyclic molecule may be substituted of
polysubstituted with C1-C5 alkyl, alkenyl, hydroxyalkyi, halogen of
haloalkyl group or with O--A or CH2--O--A wherein A is H or has the
formula CONR'R" wherein R' and R" are the same or different and are
hydrogen or a substituted or unsubstituted C1-C6 alkyl, aryl,
cycloalkyl, or heterocyclic group; or A has the formula --N--NR'R"
wherein R' and R" are the same or different and are hydrogen, or a
C1-C5 alkyl, alkenyl, hydroxyalkyl, or haloalkyl group, wherein the
halo atom is fluorine, chlorine, bromine, or iodine atom; and -M-
is an organic moiety having 1 to 10 carbon atoms and is a branched
or straight chain alkyl, aryl, or cycloalkyl group.
[0023] In one embodiment, the linker is tetraphosphopropanediol or
Pentaphosphopropanediol. In the case of polycyclic molecules there
will be preferably 18 or more atoms bridging the nucleic acids.
More preferably their will be from 30 to 50 atoms bridging, see for
Example 5. In another embodiment the linker is a bifunctional
carbazole or bifunctional carbazole linked to one or more
polyphosphoropropanediol.
[0024] Such compounds may also comprise suitable functional groups
to allow coupling through reactive groups on nucleotides."
SUMMARY OF THE INVENTION
[0025] This invention concerns the use of non-nucleotide molecules
as spacer elements at the base of double-stranded nucleic acid
(e.g., RNA or DNA) stems (duplex stems) or more preferably, in the
single-stranded regions, catalytic core, loops, or recognition arms
of enzymatic nucleic acids. Duplex stems are ubiquitous structural
elements in enzymatic RNA molecules. To facilitate the synthesis of
such stems, which are usually connected via single-stranded
nucteotide chains, a base or base-pair mimetic may be used to
reduce the nucleotide requirement in the synthesis of such
molecules, and to confer nuclease resistance (since they are
non-nucleic acid components). This also applies to both the
catalytic core and recognition arms of a ribozyme. In particular
abasic nucleotides (i.e., moieties lacking a nucleotide base, but
having the sugar and phosphate portions) can be used to provide
stability within a core of a ribozyme, e.g., at U4 or N7 or a
hammerhead structure shown in FIG. 1.
[0026] Thus, in a first aspect, the invention features an enzymatic
nucleic acid molecule having one or more non-nucleotide moieties,
and having enzymatic activity to cleave an RNA or DNA molecule.
[0027] Examples of such non-nucleotide mimetics are shown in FIG. 6
and their incorporation into hammerhead ribozymes is shown in FIG.
7. These non-nucleotide linkers may be either polyether, polyamine,
polyamide, or polyhydrocarbon compounds. Specific examples include
those described by Seela and Kaiser, Nucleic Acids Res. 1990,
18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz,
J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am.
Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Fles. 1993,
21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic
Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991, 10:287; Jaschke et at., Tetrahedron Lett. 1993,
34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al.,
International Publication No. WO 89/02439 entitled "Non-nucleotide
Linking Reagents for Nucleotide Probes"; and Ferentz and Verdine,
J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by
reference herein.
[0028] In preferred embodiments, the enzymatic nucleic acid
includes one or more stretches of RNA, which provide the enzymatic
activity of the molecule, linked to the non-nucleotide moiety.
[0029] By the term "non-nucleotide` is meant any group or compound
which can be incorporated into a nucleic acid chain in the place of
one or more nucleotide units, including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit
their enzymatic activity. The group or compound is abasic in that
it does not contain a commonly recognized nucleotide base, such as
adenine, guanine, cytosine, uracil or thymine. It may have
substitutions for a 2' or 3'H or OH as described in the art. See
Eckstein et al. and Usman et al., supra.
[0030] In preferred embodiments, the enzymatic nucleic acid
includes one or more stretches of RNA, which provide the enzymatic
activity of the molecule, linked to the non-nucleotide moiety. The
necessary ribonucleotide components are known in the art, see,
e.g., Usman, supra and Usman et al., Nucl. Acid Symp. Series,
31:163, 1994.
[0031] As the term is used in this application,
non-nucleotide-containing enzymatic nucleic acid means a nucleic
acid molecule that contains at least one non-nucleotide component
which replaces a portion of a ribozyme, e.g., but not limited to, a
double-stranded stem, a single-stranded "catalytic core" sequence,
a single-stranded loop or a single-stranded recognition sequence.
These molecules are able to cleave (preferably, repeatedly cleave)
separate RNA or DNA molecules in a nucleotide base sequence
specific manner. Such molecules can also act to cleave
intramoleculady if that is desired. Such enzymatic molecules can be
targeted to virtually any RNA transcript. Such molecules also
include nucleic acid molecules having a 3' or 5' non-nucleotide,
useful as a capping group to prevent exonuclease digestion.
[0032] Enzymatic molecules of this invention act by first binding
to a target RNA or DNA. Such binding occurs through the target
binding portion of the enzyme which is held in close proximity to
an enzymatic portion of molecule that acts to cleave the target RNA
or DNA. Thus, the molecule first recognizes and then binds a target
nucleic acid through complementary base-pairing, and once bound to
the correct site, acts enzymatically to cut the target. Strategic
cleavage of such a target will destroy its ability to direct
synthesis of an encoded protein. After an enzyme of this invention
has bound and cleaved its target it is released from that target to
search for another target, and can repeatedly bind and cleave new
targets.
[0033] The enzymatic nature of an enzyme of this invention is
advantageous over other technologies, such as antisense technology
(where a nucleic acid molecule simply binds to a nucleic acid
target to block its translation) since the effective concentration
of the enzyme necessary to effect a therapeutic treatment is lower
than that of an antisense oligonucleotide. This advantage reflects
the ability of the enzyme to act enzymatically. Thus, a single
enzyme molecule is able to cleave many molecules of target RNA. In
addition, the enzyme is a highly specific inhibitor, with the
specificity of inhibition depending not only on the base pairing
mechanism of binding, but also on the mechanism by which the
molecule inhibits the expression of the RNA to which it binds. That
is, the inhibition is caused by cleavage of the target and so
specificity is defined as the ratio of the rate of cleavage of the
targeted nucleic acid over the rate of cleavage of non-targeted
nucleic acid. This cleavage mechanism is dependent upon factors
additional to those involved in base pairing. Thus, it is thought
that the specificity of action of an enzyme of this invention is
greater than that of antisense oligonucleotide binding the same
target site.
[0034] By "complementarity" is meant a nucleic acid that can form
hydrogen bond(s) with other RNA sequence by either traditional
Watson-Crick or other non-traditional types (for example, Hoogsteen
type) of base-paired interactions.
[0035] By the phrase enzyme is meant a catalytic
non-nucleotide-containing nucleic acid molecule that has
complementarity in a substrate-binding region to a specified
nucleic acid target, and also has an enzymatic activity that
specifically cleaves RNA or DNA in that target. "That is, the
enzyme is able to intramolecularly or intermolecularly cleave RNA
or DNA and thereby inactivate a target RNA or DNA molecule. This
complementarity functions to allow sufficient hybridization of the
enzymatic molecule to the target RNA or DNA to allow the cleavage
to occur. One hundred percent complementarity is preferred, but
complementarity as low as 50-75% may also be useful in this
invention.
[0036] In preferred embodiments of this invention, the enzymatic
nucleic acid molecule is formed in a hammerhead or hairpin motif,
but may also be formed in the motif of a hepatitis delta virus,
group I intron or RNaseP RNA (in association with an RNA guide
sequence) or Neurospora VS RNA. Examples of such hammerhead motifs
are described by Rossi et al., 1992, Aids Research and Human
Retroviruses 8, 183, of hairpin motifs by Hampei et al., EP0360257,
Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al.,
1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis
delta virus motif is described by Perrotta and Been, 1992
Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al.,
1983 Cell 35, 849, Neurospora VS RNA ribozyme motif is described by
Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and
Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and
Olive, 1993 Biochemistry 32, 2795-2799) and of the Group I intron
by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are
not limiting in the invention and those skilled in the art will
recognize that all that is important in an enzymatic nucleic acid
molecule of this invention is that it has a specific substrate
binding site which is complementary to one or more of the target
gene RNA regions, and that it have nucleotide sequences within or
surrounding that substrate binding site which impart an RNA
cleaving activity to the molecule.
[0037] The invention provides a method for producing a class of
enzymatic cleaving agents which exhibit a high degree of
specificity for the nucleic acid of a desired target. The enzyme
molecule is preferably targeted to a highly conserved sequence
region of a target such that specific treatment of a disease or
condition can be provided with a single enzyme. Such enzyme
molecules can be delivered exogenously to specific cells as
required. In the preferred hammerhead motif the small size (less
than 60 nucleotides, preferably between 30-40 nucleotides in
length) of the molecule allows the cost of treatment to be reduced
compared to other ribozyme motifs.
[0038] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods, and the therapeutic
cost of such molecules is prohibitive. In this invention, small
enzyme motifs (e.g., of the hammerhead structure) are used for
exogenous delivery. The simple structure of these molecules
increases the ability of the enzyme to invade targeted regions of
mRNA structure. Unlike the situation when the hammerhead structure
is included within longer transcripts, there are no non-enzyme
flanking sequences to interfere with correct folding of the enzyme
structure or with complementary regions.
[0039] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a diagrammatic representation of a hammerhead
ribozyme domain known in the art. Stem II can be >2 base-pair
long, or can even lack base pairs and consist of a loop region.
[0041] FIG. 2a is a diagrammatic representation of the hammerhead
ribozyme domain known in the art; FIG. 2b is a diagrammatic
representation of the hammerhead ribozyme as divided by Uhlenbeck
(1987, Nature, 327, 596) into a substrate and enzyme portion; FIG.
2c is a similar diagram showing the hammerhead divided by Haseloff
and Gertach (1988, Nature, 334, 585) into two portions; and FIG. 2d
is a similar diagram showing the hammerhead divided by Jeffries and
Symons (1989, Nucleic. Acids. Res., 17, 1371) into two
portions.
[0042] FIG. 3 is a diagrammatic representation of the general
structure of a hairpin ribozyme. Helix 2 (H2) is provided with a
least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be
optionally provided of length 2 or more bases (preferably 3-20
bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 may be
covalently linked by one or more bases (i.e., r is >1 base).
Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g.,
4 - 20 base pairs) to stabilize the ribozyme structure, and
preferably is a protein binding site. In each instance, each N and
N' independently is any normal or modified base and each dash
represents a potential base-pairing interaction. These nucleotides
may be modified at the sugar, base or phosphate. Complete
base-pairing is not required in the helices, but is preferred.
Helix 1 and 4 can be of any size (i.e., o and p is each
independently from 0 to any number, e.g., 20) as long as some
base-pairing is maintained. Essential bases are shown as specific
bases in the structure, but those in the art will recognize that
one or more may be modified chemically (abasic, base, sugar and/or
phosphate modifications) or replaced with another base without
significant effect. Helix 4 can be formed from two separate
molecules, i.e., without a connecting loop. The connecting loop
when present may be a dbonucleotide with or without modifications
to its base, sugar or phosphate. "q" is >2 bases. The connecting
loop can also be replaced with a non-nucleotide linker molecule. H,
refers to bases A, U or C. Y refers to pyrimidine bases. "--"
refers to a chemical bond.
[0043] FIG. 4 is a representation of the general structure of the
hepatitis delta virus ribozyme domain known in the art (Perrota and
Been, 1991 supra).
[0044] FIG. 5 A is a representation of the general structure of the
self-cleaving Neurospora VS RNA domain. 5B is a line diagram
representing the "1" ribozyme motif. The figure shows the "Upper
and the "Lower" base-paired regions linked by the "connecting"
region. IV (left) and V (right) shows the left and the right handed
regions within the "upper" region, respectively. II (left) and Vl
(right) shows the left and the right handed regions within the
"lower" region, respectively).
[0045] FIG. 6 is a diagrammatic representation of various
non-nucleotide mimetics that may be incorporated into nucleic acid
enzymes. Standard abbreviations are used in the Figure. In compound
1 each X may independently be oxygen, nitrogen, sulfur or
substituted carbons containing elkyl, alkene or equivalent chains
of length 1-10 carbon atoms. In compounds 6, 6a, 7, 8, 9 and 10
each Y may independently be a phosphodiester, ether or amide
linkage to the rest of the nucleic acid enzyme. In compounds 4 and
5 each R may independently be H, OH, protected OH, O-alkyl, alkenyl
or alkynyl or alkyl, alkenyl or alkynyl of 1 -10 carbon atoms.
[0046] FIG. 7 is a diagrammatic representation of the preferred
location for incorporation of various non-nucleotide mimetics into
nucleic acid enzymes. Specifically, mimetics, 1-10, may replace the
loop (denoted as / / in FIG. 7) that connects the two strands of
Stem II Stem II itself may be from 1 to 10 base pairs. In examples
1 & 2 below compounds 1 and 2 were incorporated into molecules
having a stem II of 1 to 5 basepairs in length. Compounds 1,4 and 5
may also replace nucleotides in the recognition arms of stems I and
III or in stem II itself.
[0047] FIG. 8 is a diagrammatic representation of the synthesis of
a perylene based non-nucleotide mimetic phosphoramidite 3.
[0048] FIGS. 9A and 9B are a diagrammatic representation of the
synthesis of an abasic deoxyribose or ribose non-nucleotide mimetic
phosphoramidite.
[0049] FIGS. 10A and 10B are graphical representations of cleavage
of substrate by various ribozymes at 8 nM, or 40 nM,
respectively.
[0050] FIG. 11 is a diagrammatic representation of a hammerhead
ribozyme targeted to site A (HHA). Arrow indicates the cleavage
site. Stem II is shorter than usual for a hammerhead ribozyme.
[0051] FIG. 12 is a diagrammatic representation of HHA ribozyme
containing abasic substitutions (HHA-a) at various positions.
Ribozymes were synthesized as described in the application. "X"
shows the positions of abasic substitutions. The abasic
substitutions were either made individually or in certain
combinations.
[0052] FIG. 13 shows the in vitro RNA cleavage activity of HHA and
HHA-a ribozymes. All RNA, refers to HHA ribozyme containing no
abasic substitution. U4 Abasic, refers to HHA-a ribozyme with a
single abasic (ribose) substitution at position 4. U7 Abasic,
refers to HHA-a ribozyme with a single abasic (ribose) substitution
at position 7.
[0053] FIG. 14 shows in vitro RNA cleavage activity of HHA and
HHA-a ribozymes. Abasic Stem II Loop, refers to HHA-a ribozyme with
four abasic (ribose) substitutions within the loop in stem II.
[0054] FIG. 15 shows in vitro RNA cleavage activity of HHA and
HHA-a ribozymes. 3'-Inverted Deoxyribose, refers to HHA-a ribozyme
with an inverted deoxyribose (abasic) substitution at its 3'
termini.
[0055] FIG. 16 is a diagrammatic representation of a hammerhead
ribozyme targeted to site B (HHB). Target B is involved in the
proliferation of mammalian smooth muscle cells. Arrow indicates the
site of cleavage. Inactive version of HHB contains 2
base-substitutions (G5U and A15.1U) that renders the ribozyme
catalytically inactive.
[0056] FIG. 17 is a diagrammatic representation of HHB ribozyme
with abasic substitution (HHB-a) at position 4. X, shows the
position of abasic substitution.
[0057] FIG. 18 shows ribozyme-mediated inhibition of rat aortic
smooth muscle cell (RASMC) proliferation. Both HHB and HHB-a
ribozymes can inhibit the proliferation of RASMC in culture.
Catalytically inactive HHB ribozyme shows inhibition which is
significantly lower than active HHB and HHB-a ribozymes.
DETAILED DESCRIPTION OF THE INVENTION
Non-nucleotide Mimetics
[0058] Non-nucleotide mimetics useful in this invention are
generally described above. Those in the art will recognize that
these mimetics can be incorporated into an enzymatic molecule by
standard techniques at any desired location. Suitable choices can
be made by standard experiments to determine the best location,
e.g., by synthesis of the molecule and testing of its enzymatic
activity. The optimum molecule will contain the kIown
dbonucleotides needed for enzymatic activity, and will have
non-nucleotides which change the structure of the molecule in the
least way possible. What is desired is that several nucleotides can
be substituted by one non-nucleotide to save synthetic steps in
enzymatic molecule synthesis and to provide enhanced stability of
the molecule compared to RNA or even DNA.
Synthesis of Ribozymes
[0059]
[0060] In this invention, small enzymatic nucleic acid motifs
(e.g., of the hammerhead or the hairpin structure) are used for
exogenous delivery. The simple structure of these molecules
increases the ability of the enzymatic nucleic acid to invade
targeted regions of the mRNA structure. The ribozymes are
chemically synthesized. The method of synthesis used follows the
procedure for normal RNA synthesis as described in Usman et al.,
1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990
Nucleic Acids Res., 18, 5433 and makes use of common nucleic acid
protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosphoramidites at the 3'-end. The average stepwise
coupling yields were >98%.
[0061] Ribozymes are purified by gel electrophoresis using general
methods or are purified by high pressure liquid chromatography
(HPLC; See Usman et al., Synthesis, deprotection, analysis and
purification of RNA and ribozymes, filed May, 18, 1994, U.S. Ser.
No. 08/245,736 the totality of which is hereby incorporated herein
by reference) and are resuspended in water.
[0062] Various modifications to ribozyme structure can be made to
enhance the utility of ribozymes. Such modifications will enhance
shelf-life, half-life in vitro, stability, and ease of introduction
of such ribozymes to the target site, e.g., to enhance penetration
of cellular membranes, and confer the ability to recognize and bind
to targeted cells.
Optimizing Ribozyme Activity
[0063] Ribozyme activity can be optimized as described by
Stinchcomb et al., "Method and Composition for Treatment of
Restenosis and Cancer Using Ribozymes," filed May 18, 1994, U.S.
Ser. No. 08/245,466. The details will not be repeated here, but
include altering the length of the ribozyme binding arms (stems I
and III, see FIG. 2c), or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases
(see e.g., Eckstein et al., International Publication No. WO
92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al.,
1991 Science 2S3, 314; Usman and Cedergren, 1992 Trends in Biochem.
Sci. 17, 334; Usman et al., International Publication No. WO
93/15187; and Rossi et al., International Publication No. WO
91/03162, as well as Usman, N. et al. U.S. patent application Ser.
No. 07/829,729, and Sproat, European Patent Application 92110298.4
which describe various chemical modifications that can be made to
the sugar moieties of enzymatic RNA molecules. Modifications which
enhance their efficacy in cells, and removal of stem II bases to
shorten RNA synthesis times and reduce chemical requirements (All
these publications are hereby incorporated by reference
herein).
Administration of Ribozyme
[0064] Sullivan et al., PCT WO94/02595, describes the general
methods for delivery of enzymatic RNA molecules. Ribozymes may be
administered to cells by a variety of methods known to those
familiar to the art, including, but not restricted to,
encapsulation in liposomes, by iontophoresis, or by incorporation
into other vehicles, such as hydrogels, cyclodextrins,
biodegradable nanocapsules, and bioadhesive microspheres. For some
indications, ribozymes may be directly delivered ex vivo to cells
or tissues with or without the aforementioned vehicles.
Alternatively, the RNA/vehicle combination is locally delivered by
direct injection or by use of a catheter, infusion pump or stent.
Other routes of delivery include, but are not limited to,
intravascular, intramuscular, subcutaneous or joint injection,
aerosol inhalation, oral (tablet or pill form), topical, systemic,
ocular, intraperitoneal and/or intrathecal delivery. More detailed
descriptions of ribozyme delivery and administration are provided
in Sullivan et al., supra and Draper et al., PCT WO93/23569 which
have been incorporated by reference herein.
EXAMPLES
[0065] The following are non-limiting examples showing the
synthesis of non-nucleotide mimetic-containing catalytic nucleic
acids using non-nucleotide phosphoramidites.
[0066] As shown in FIG. 7, such non-nucleotides can be located in
the binding arms, core or the loop adjacent stem II of a hammerhead
type ribozyme. Those in the art following the teachings herein can
determine optimal locations in these regions. Surprisingly, abasic
moieties can be located in the core of such a ribozyme.
Example 1
Synthesis of Hammerhead Ribozymes Containing Non-nucleotide
Mimetics: Polyether Spacers
[0067] Polyether spacers, compound I (FIG. 6; X=O, n=2 or 4), have
been incorporated both singly, n=2 or 4, or doubly, n=2, at the
base of stem II of a hammerhead ribozyme, replacing loop 2, and
shown to produce a ribozyme which has lower catalytic efficiency.
The method of synthesis used followed the procedure for normal RNA
synthesis as described in Usman et al., J. Am. Chem. Soc. 1987,
109:7845 and in Scaringe et al., Nucleic Acids Res. 1990, 18:5433,
and makes use of common nucleic acid protecting and coupling
groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites
at the 3'-end. The average stepwise coupling yields were >98%.
The design of these types of mimetics has not been optimized to
date, but, as discussed above, this can be readily achieved using
standard experimental techniques. These experiments indicate the
potential of such mimetics to replace the loops and portions of
stems in ribozymes while maintaining catalytic activity. These
mimetics may be incorporated not only into hammerhead ribozymes,
but also into hairpin, hepatitis delta virus, or Group 1 or Group 2
introns. They are, therefore, of general use as replacement motifs
in any nucleic acid structure. Use of such mimetics allows about
2-10 nucleotides to be omitted from the final nucleic acid molecule
compared to the use of an oligonucleotide without a non-nucleotide
mimetic.
Example 2
Synthesis of Hammerhead Ribozymes Containing Non-nucleotide
Mimetics: Aromatic Spacers
[0068] In another example, a specific linker for the base of the
stem II C-G of a hammerhead ribozyme was designed. Applicant
believes that the distance between the C1' carbons of the C-G base
pair is about 16 Angstroms. To join these two pieces of RNA by a
covalent analog of the C-G base pair a new type of dimer
phosphoramidite containing a linker between the 3'-OH and the 5'-OH
of the G and C residues respectively can be constructed. Two types
of base-pair mimetic are the rigid aromatic spacers, 2 or 3, shown
in FIG. 6. These have been incorporated at the base of stem II of a
hammerhead ribozyme as described in Example 1, replacing loop 2,
and shown to produce a ribozyme which has lower catalytic
efficiency. Another mimetic is a flexible alkyl spacer similar to
the polyamide backbone described by Nielsen et al., Science 1991,
254:1497 (see, FIG. 6; 6 or a derivative thereof 6a; Zuckerman et
al., J. Am. Chem. Soc. 1992, 114:10646). Use of such mimetics
allows about 2-10 nucleotides to be omitted from the final nucleic
acid molecule compared to the use of an oligonucleotide without a
non-nucleotide mimetic.
Example 3
Synthesis of Non-nucleotide Mimetics Aromatic Spacer
Phosphoramidite 2
[0069] This compound was originally described by Salunkhe et al.,
J. Am. Chem. Soc. 1992, 114:8768. The synthesis was modified as
follows: To terphthalic acid (1.0 g, 6.0 retool) in DMF (12 mL) was
added EDC (2.54 g, 13.2 mmol), aminohexanol (1.55 g, 13.2 mmol) and
N-methylmorpholine (1.45 mL, 13.2 mmol). The reaction mixture was
stirred overnight at which time the solution was cloudy. Water was
added to the reaction mixture to precipitate out the product. The
solid was filtered and washed with water and dried to provide 562
mg (25.7%) of the diol.
[0070] To the diol (250 mg, 0.687 mmol) in DMSO (40 mL) was added
triethylamine (287 .mu.L, 2.06 mmol), dimethoxytrityl chlodde (220
mg, 0.653 mmol) and catalytic DMAP. The reaction mixture was heated
to 40.degree. C. and stirred overnight. The mixture was then cooled
to room temperature (about 20-25.degree. C.), quenched with water
and extracted three times with EtOAc. A solid precipitate remained
in the organic layer that was isolated and found to be starting
diol (50 mg, 20%). The organic layer was dried over Na2SO4 and
evaporated. The resulting oil was purified with flash
chromatography (10% EtOAc in hexanes to 100% EtOAc) to yield 250 mg
(55%) of the monotritylated compound.
[0071] To the alcohol (193 mg, 0.29 mmol) in THF (1 mL) at
0.degree. C. was added diisopropylethylamine (101 .mu.L, 0.58 mmol)
and then 2-cyanoethyl N,N-diisopropylamino chlorophosphoramidite
(78 .mu.L, 0.35 mmol) dropwise. The resulting mixture was stirred
for 5 minutes and then warmed to room temperature. After 1 hour the
reaction mixture was quenched with methanol and evaporated. The
resulting oil was purified by flash chromatography (1:1
hexanes:EtOAc) to yield 158 mg (63%) of the phosphoramidite.
Example 4
Synthesis of Non-nucleotide Mimetics Aromatic Spacer
Phosphoramidite 3
[0072] Referring to FIG. 8, to 3, 4, 9, 10-perylenetetracarboxylic
dianhydride 11 (1.0 g, 2.55 mmol) in quinoline (10 mL) was added
ethanolamine (919 .mu.L, 15.3 mmol) and ZnOAc.2.5 H20 (140 mg,
0.638 mmol). The reaction mixture was heated to 190.degree. C. for
8 hours. The solution was then cooled, 1N HCI added to precipitate
the product and the mixture was filtered. The solid was washed with
hot 10% CaCO3 until the filtrate was no longer pale green. The
remaining bright red precipitate 12 was then dried.
[0073] The resulting diol 12 was then treated as outlined above for
2 to provide the phosphoramidite 3.
Example 5
Synthesis of Hammerhead Ribozymes Containing Non-nucleotide
Mimetics: Abasic Nucleotides 4 and 5
[0074] Compound 4, R.dbd.H, was prepared according to Iyer et al.,
Nucleic Acids Res. 1990, 18:2855. Referring to FIG. 9A, compounds 4
and 5 (R.dbd.O-t-butytdimethylsilyl) phosphoramidites were prepared
as follows:
[0075] To a solution of D-ribose (20.0 g, 0.105 tool) in
N,N-dimethylformamide (250 mL) was added 2,2-dimethoxypropane (50
mL) and p-toluenesulfonic acid monohydrate (300 mg). The reaction
mixture was stirred for 16 hours at room temperature and then
evaporated to dryness. The crude product was coevaporated with
pyridine (2.times.150 mL), dissolved in dry pyfidine (300 mL) and
4,4'-dimethoxytrityl chloride (37.2 g, 0.110 mol) was added and
stirred for 24 hours at room temperature. The reaction mixture was
diluted with methanol (50 mL) and evaporated to dryness. The
residue was dissolved in chloroform (800 mL) and washed with 5%
NaHCO3 (2.times.200 mL), brine (300 mL), dried, evaporated,
coevaporated with toluene (2.times.100 mL) and purified by flash
chromatography in CHCI3 to yield 40.7 g (78.1%) of compound a.
[0076] To a solution of dimethoxytrityl derivative a (9.0 g, 18.3
mmol) and DMAP (4.34 g, 36 mmol) in dry CH3CN, phenoxythiocarbonyl
chloride (3.47 g, 20.1 mmol) was added dropwise under argon. The
reaction mixture was left for 16 hours at room temperature, then
evaporated to dryness. The resulting residue was dissolved in
chloroform (200 mL), washed with 5% NaHCO3, brine, dried,
evaporated and purified by flash chromatography in CHCl3, to yield
8.0 g (69.5%) of compound b as the .beta.-anomer.
[0077] To a solution of intermediate b (3.0 g, 4.77 mmol) in
toluene (50 mL) was added AIBN (0.82 g, 5.0 mmol) and Bu3SnH (1.74
g, 6.0 mmol) under argon and the reaction mixture was kept at
80.degree. C. for 7 hours. The solution was evaporated and the
resulting residue purified by flash chromatography in CHCI3 to
yield 1.5 g (66%) of protected ribitol c.
[0078] Subsequent removal of all protecting groups by acid
treatment and tritylation provided the protected ribitol d which
was then converted to target phosphoramidites 4 and 5 by the
general method described in Scaringe et al., Nucleic Acids Res.
1990, 18:5433.
[0079] The synthesis of 1-deoxy-D-ribofuranose phosphoramidite 9 is
shown in FIG. 9B. Our initial efforts concentrated on the
deoxygenation of synthon 1, prepared by a "one pot" procedure from
D-ribose. Phenoxythiocarbonylation of acetonide 1 under Robins
conditions led to the .beta.-anomer 2 (J1,2=1.2 Hz) in modest yield
(45-55%). Radical deoxygenation using Bu3SnH/AIBN resulted in the
formation of the ribitol derivative 3 in 50% yield. Subsequent
deprotection with 90% CF3COOH (10 m) and introduction of a
dimethoxytrityl group led to the key intermediate 4 in 40% yield
(Yang et al., Biochemistry 1992, 31, 5005-5009; Perreault et al.,
Biochemistry 1991, 30, 4020-4025; Paolella et al., EMBO J. 1992,
11, 1913-1919; Peiken et al., Science 1991, 253, 314-317).
[0080] The low overall yield of this route (FIG. 9A) prompted us to
investigate a different approach to 4 (FIG. 9B).
Phenylthioglycosides, successfully employed in the Keck reaction,
appeared to be an alternative. However, it is known that
free-radical reduction of the corresponding glycosyl bromides with
participating acyl groups at the C2-position can result in the
migration of the 2-acyl group to the C1-position (depending on
Bu3SnH concentration). Therefore we subjected phenytthioglycoside 5
to radical reduction with Bu3SnH (6.1 eq.) in the presence of Bz202
(2 eq.) resulting in the isolation of tribenzoate 6 in 63% yield
(FIG. 9B). Subsequent debenzoylation and dimethoxytritylation led
to synthon 4 in 70% yield. Introduction of the TBDMS group, using
standard conditions, resulted in the formation of a 4:1 ratio of 2-
and 3-isomers 8 and 7. The two regioisomers were separated by
silica gel chromatography. The 2-O-t-butyldimethylsilyl derivative
8 was phosphitylated to provide phosphoramidite 9 in 82% yield.
Example 6
[0081] Referring to FIGS. 10a and 10b the cleavage of substrate is
shown by various modified ribozymes compared to unmodified ribozyme
at 8 nM and 40 nM concentrations. Specifically, a control ribozyme
of sequence ucuccA UCU GAU GAG GCC GAA AGG CCG AAA Auc ccU (SEQ ID
NO. 17) (where lower case includes a 2'O-methyl group) was compared
to ribozyme A (ucu ccA UCU GAU GAG GCC SGG CCG AAA Auc ccu (SEQ ID
NO. 18)), B (ucu ccA UCU GAU GAG CSG CG AAA Auc ccu (SEQ ID NO.
19)), C (ucu ccA UCU GAU GAG GCC bbb bGG CCG AAA Auc ccu (SEQ ID
NO. 20)), and D (ucu ccA UCU GAU GAG Cbb bbG CGAA A.Au ccc u (SEQ
ID NO. 21)) (where S=hexaethylene glycol linker); and b=abasic
nucleotide 4). All were active in cleaving substrate.
Example 7
RNA Cleavage Assay in vitro
[0082] Ribozymes and substrate RNAs were synthesized as described
above. Substrate RNA was 5' end-labeled using [.gamma.-32p] ATP and
T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were
carried out under ribozyme "excess" conditions. Trace amount (<1
nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme were
denatured and renatured separately by heating to 90.degree. C. for
2 min and snap-cooling on ice for 10-15 min. The ribozyme and
substrate were incubated, separately, at 37.degree. C. for 10 min
in a buffer containing 50 mM Tris-HCI and 10 mM MgCI2. The reaction
was initiated by mixing the ribozyme and substrate solutions and
incubating at 37.degree. C. Aliquots of 5 .mu.l are taken at
regular intervals of time and the reaction quenched by mixing with
an equal volume of 2.times. formamide stop mix. The samples were
resolved on 20% denaturing polyacrylamide gels. The results were
quantified and percentage of target RNA cleaved is plotted as a
function of time.
[0083] Referring to FIG. 11 there is shown the general structure of
a hammerhead ribozyme targeted against site A (HHA) with various
bases numbered. Various substitutions were made at several of the
nucleotide positions in HHA. Specifically referring to FIG. 12,
substitutions were made at the U4 and U7 positions marked as X4 and
X7 and also in loop II in the positions marked by an X. The RNA
cleavage activity of these substituted ribozymes is shown in the
following figures. Specifically, FIG. 13 shows cleavage by an
abasic substituted U4 and an abasic substituted U7. As will be
noted, abasic substitution at U4 or U7 does not significantly
affect cleavage activity. In addition, inclusion of all abasic
moieties in stem II loop does not significantly reduce enzymatic
activity as shown in FIG. 14. Further, inclusion of a 3' inverted
deoxyribos does not inactivate the RNA cleavage activity as shown
in FIG. 15.
Example 8
Smooth Muscle Cell Proliferation Assay
[0084] Hammerhead ribozyme (HHB) is targeted to a unique site (site
B) within c-myb mRNA. Expression of c-myb protein has been shown to
be essential for the proliferation of rat smooth muscle cell (Brown
et al., 1992 J. Biol. Chem. 267, 4625).
[0085] The ribozymes that cleaved site B within c-myb RNA described
above were assayed for their effect on smooth muscle cell
proliferation. Rat vascular smooth muscle cells were isolated and
cultured as described (Stnchcomb et al., supra). These primary rat
aortic smooth muscle cells (RASMC) were plated in a 24-well plate
(5.times.103 cells/well) and incubated at 37.degree. C. in the
presence of Dulbecco's Minimal Essential Media (DMEM) and 10% serum
for -16 hours.
[0086] These cells were serum-starved for 48-72 hours in DMEM
(containing 0.5% serum) at 37.degree. C. Following
serum-starvation, the cells were treated with lipofectamine
(LFA)-complexed ribozymes (100 nM ribozyme was complexed with LFA
such that LFA:ribozyme charge ration is 4:1).
[0087] Ribozyme:LFA complex was incubated with serum-starved RASMC
cells for four hours at 37.degree. C. Following the removal of
ribozyme:LFA complex from cells (after 4 hours), 10% serum was
added to stimulate smooth cell proliferation. Bromo-deoxyuddine
(BrdU) was added to stain the cells. The cells were stimulated with
serum for 24 hours at 37.degree. C.
[0088] Following serum-stimulation, RASMC cells were quenched with
hydrogen peroxide (0.3% H2O2 in methanol) for 30 min at 4.degree.
C. The cells were then denatured with 0.5 ml 2N HCI for 20 min at
room temperature. Horse serum (0.5 ml) was used to block the cells
at 4.degree. C. for 30 min up to -16 hours.
[0089] The RASMC cells were stained first by treating the cells
with anti-BrdU (primary) antibody at room temperature for 60 min.
The cells were washed with phosphate-buffered saline (PBS) and
stained with biotinylated affinity-purified anti-mouse IgM (Pierce,
USA) secondary antibody. The cells were counterstained using
avidin-biotinylated enzyme complex (ABC) kit (Pierce, USA).
[0090] The ratio of proliferating:non-proliferating cells was
determined by counting stained cells under a microscope.
Proliferating RASMCs will incorporate BrdU and will stain brown.
Non-proliferating cells do not incorporate BrdU and will stain
purple.
[0091] Referring to FIG. 16 there is shown a ribozyme which cleaves
the site B referred to as HHB. Substitutions of abasic moieties in
place of U4 as shown in FIG. 17 provided active ribozyme as shown
in FIG. 18 using the above-noted rat aortic smooth muscle cell
proliferation assay.
Administration of Ribozyme
[0092] Selected ribozymes can be administered prophylactically, to
viral infected patients or to diseased patients, e.g., by exogenous
delivery of the ribozyme to a relevant tissue by means of an
appropriate delivery vehicle, e.g., a liposome, a controlled
release vehicle, by use of iontophoresis, electroporation or ion
paired molecules, or covalently attached adducts, and other
pharmacologically approved methods of delivery. Routes of
administration include intramuscular, aerosol, oral (tablet or pill
form), topical, systemic, ocular, intraperitoneal and/or
intrathecal.
[0093] The specific delivery route of any selected ribozyme will
depend on the use of the ribozyme. Generally, a specific delivery
program for each ribozyme will focus on unmodified ribozyme uptake
with regard to intracellular localization, followed by
demonstration of efficacy. Alternatively, delivery to these same
cells in an organ or tissue of an animal can be pursued. Uptake
studies will include uptake assays to evaluate cellular ribozyme
uptake, regardless of the delivery vehicle or strategy. Such assays
will also determine the intracellular localization of the ribozyme
following uptake, ultimately establishing the requirements for
maintenance of steady-state concentrations within the cellular
compartment containing the target sequence (nucleus and/or
cytoplasm). Efficacy and cytotoxicity can then be tested. Toxicity
will not only include cell viability but also cell function.
[0094] Some methods of delivery that may be used include:
[0095] a. encapsulation in liposomes,
[0096] b. transduction by retroviral vectors,
[0097] c. conjugation with cholesterol,
[0098] d. localization to nuclear compartment utilizing antigen
binding or nuclear targeting site found on most snRNAs or nuclear
proteins,
[0099] e. neutralization of charge of ribozyme by using nucleotide
derivatives, and
[0100] f. use of blood stem cells to distribute ribozymes
throughout the body.
[0101] Delivery strategies useful in the present invention,
include: ribozyme modifications, and particle carrier drug delivery
vehicles. Unmodified ribozymes, like most small molecules, are
taken up by cells, albeit slowly. To enhance cellular uptake, the
ribozyme may be modified essentially at random, in ways which
reduce its charge but maintains specific functional groups. This
results in a molecule which is able to diffuse across the cell
membrane, thus removing the permeability bamrer.
[0102] Modification of ribozymes to reduce charge is just one
approach to enhance the cellular uptake of these larger molecules.
The random approach, however, is not advisable since ribozymes are
structurally and functionally more complex than small drug
molecules. The structural requirements necessary to maintain
ribozyme catalytic activity are well understood by those in the
art. These requirements are taken into consideration when designing
modifications to enhance cellular delivery. The modifications are
also designed to reduce susceptibility to nuclease degradation.
Both of these characteristics should greatly improve the efficacy
of the ribozyme. Cellular uptake can be increased by several orders
of magnitude without having to alter the phosphodiester linkages
necessary for ribozyme cleavage activity.
Use
[0103] Those in the art will recognize that these ribozymes can be
used in place of other enzymatic RNA molecules for both in vitro
and in vivo uses well known in the art. See Draper WO 93/23569 and
Sullivan WO 94/12516.
[0104] Chemical modifications of the phosphate backbone will reduce
the negative charge allowing free diffusion across the membrane.
This principle has been successfully demonstrated for antisense DNA
technology. The similarities in chemical composition between DNA
and RNA make this a feasible approach. In the body, maintenance of
an external concentration will be necessary to drive the diffusion
of the modified ribozyme into the cells of the tissue.
Administration routes which allow the diseased tissue to be exposed
to a transient high concentration of the drug, which is slowly
dissipated by systemic adsorption are preferred. Intravenous
administration with a drug carrier designed to increase the
circulation half-life of the ribozyme can be used. The size and
composition of the drug carrier restricts rapid clearance from the
blood stream. The carrier, made to accumulate at the site of
infection, can protect the ribozyme from degradative processes.
[0105] Drug delivery vehicles are effective for both systemic and
topical administration. They can be designed to serve as a slow
release reservoir, or to deliver their contents directly to the
target cell. An advantage of using direct delivery drug vehicles is
that multiple molecules are delivered per uptake. Such vehicles
have been shown to increase the circulation half-life of drugs
which would otherwise be rapidly cleared from the blood stream.
Some examples of such specialized drug delivery vehicles which fall
into this category are liposomes, hydrogels, cyclodextrins,
biodegradable nanocapsules, and bioadhesive microspheres.
[0106] From this category of delivery systems, liposomes are
preferred. Liposomes increase intracellular stability, increase
uptake efficiency and improve biological activity.
[0107] Liposomes are hollow spherical vesicles composed of lipids
arranged in a similar fashion.as those lipids which make up the
cell membrane. They have an internal aqueous space for entrapping
water soluble compounds and range in size from 0.05 to several
microns in diameter. Several studies have shown that liposomes can
deliver RNA to cells and that the RNA remains biologically
active.
[0108] For example, a liposome delivery vehicle originally designed
as a research tool, Lipofectin, has been shown to deliver intact
mRNA molecules to cells yielding production of the corresponding
protein. In another study, an antibody targeted liposome delivery
system containing an RNA molecule 3,500 nucleotides in length and
antisense to a structural protein of HIV, inhibited virus
proliferation in a sequence specific manner. Not only did the
antibody target the liposomes to the infected cells, but it also
triggered the internalization of the liposomes by the infected
cells. Triggering the endocytosis is useful for viral inhibition.
Finally, liposome delivered synthetic ribozymes have been shown to
concentrate in the nucleus of H9 (an example of an HIV-sensitive
cell) cells and are functional as evidenced by their intracellular
cleavage of the sequence. Liposome delivery to other cell types
using smaller ribozymes (less than 142 nucleotides in length)
exhibit different intracellular localizations.
[0109] Liposomes offer several advantages: They are non-toxic and
biodegradable in composition; they display long circulation
half-lives; and recognition molecules can be readily attached to
their surface for targeting to tissues. Finally, cost effective
manufacture of liposome-based pharmaceuticals, either in a liquid
suspension or lyophilized product, has demonstrated the viability
of this technology as an acceptable drug delivery system.
[0110] Other controlled release drug delivery systems, such as
nonoparticles and hydrogels may be potential delivery vehicles for
a ribozyme. These carriers have been developed for chemotherapeutic
agents and protein-based pharmaceuticals, and consequently, can be
adapted for ribozyme delivery.
[0111] Topical administration of ribozymes is advantageous since it
allows localized concentration at the site of administration with
minimal systemic adsorption. This simplifies the delivery strategy
of the ribozyme to the disease site and reduces the extent of
toxicological characterization. Furthermore, the amount of material
to be applied is far less than that required for other
administration routes. Effective delivery requires the ribozyme to
diffuse into the infected cells. Chemical modification of the
ribozyme to neutralize negative charge may be all that is required
for penetration. However, in the event that charge neutralization
is insufficient, the modified ribozyme can be co-formulated with
permeability enhancers, such as Azone or oleic acid, in a liposome.
The liposomes can either represent a slow release presentation
vehicle in which the modified ribozyme and permeability enhancer
transfer from the liposome into the infected cell, or the liposome
phospholipids can participate directly with the modified ribozyme
and permeability enhancer in facilitating cellular delivery. In
some cases, both the ribozyme and permeability enhancer can be
formulated into a suppository formulation for slow release.
[0112] Ribozymes may also be systematically administered. Systemic
absorption refers to the accumulation of drugs in the blood stream
followed by distribution throughout the entire body. Administration
routes which lead to systemic absorption include: intravenous,
subcutaneous, intraperitoneal, intranasal, intrathecal and
ophthalmic. Each of these administration routes expose the ribozyme
to an accessible diseased tissue. Subcutaneous administration
drains into a localized lymph node which proceeds through the
lymphatic network into the circulation. The rate of entry into the
circulation has been shown to be a function of molecular weight or
size. The use of a liposome or other drug carrier localizes the
ribozyme at the lymph node. The ribozyme can be modified to diffuse
into the cell, or the liposome can directly participate in the
delivery of either the unmodified or modified ribozyme to the cell.
This method is particularly useful for treating AIDS using anti-HIV
ribozymes.
[0113] Also preferred in AIDS therapy is the use of a liposome
formulation which can deliver oligonucleotides to lymphocytes and
macrophages. This oligonucleotide delivery system inhibits HIV
proliferation in infected primary immune cells. Whole blood studies
show that the formulation is taken up by 90% of the lymphocytes
after 8 hours at 37.degree. C. Preliminary biodistribution and
pharmacokinetic studies yielded 70% of the injected dose/gm of
tissue in the spleen after one hour following intravenous
administration. This formulation offers an excellent delivery
vehicle for anti-AIDS ribozymes for two reasons. First, T-helper
lymphocytes and macrophages are the primary cells infected by the
virus, and second, a subcutaneous administration delivers the
ribozymes to the resident HIV-infected lymphocytes and macrophages
in the lymph node. The liposomes then exit the lymphatic system,
enter the circulation, and accumulate in the spleen, where the
ribozyme is delivered to the resident lymphocytes and
macrophages.
[0114] Intraperitoneal administration also leads to entry into the
circulation, with once again, the molecular weight or size of the
ribozyme-delivery vehicle complex controlling the rate of
entry.
[0115] Liposomes injected intravenously show accumulation in the
liver, lung and spleen. The composition and size can be adjusted so
that this accumulation represents 30% to 40% of the injected dose.
The remaining dose circulates in the blood stream for up to 24
hours.
[0116] The chosen method of delivery should result in cytoplasmic
accumulation in the afflicted cells and molecules should have some
nuclease-resistance for optimal dosing. Nuclear delivery may be
used but is less preferable. Most preferred delivery methods
include liposomes (10-400 nm), hydrogels, controlled-release
polymers, microinjection or electroporation (for ex vivo
treatments) and other pharmaceutically applicable vehicles. The
dosage will depend upon the disease indication and the route of
administration but should be between 100-200 mg/kg of body
weight/day. The duration of treatment will extend through the
course of the disease symptoms, usually at least 14-16 days and
possibly continuously. Multiple daily doses are anticipated for
topical applications, ocular applications and vaginal applications.
The number of doses will depend upon disease delivery vehicle and
efficacy data from clinical trials.
[0117] Establishment of therapeutic levels of ribozyme within the
cell is dependent upon the rate of uptake and degradation.
Decreasing the degree of degradation will prolong the intracellular
half-life of the ribozyme. Thus, chemically modified ribozymes,
e.g., with modification of the phosphate backbone, or capping of
the 5' and 3' ends of the ribozyme with nucleotide analogues may
require different dosaging. Descriptions of useful systems are
provided in the art cited above, all of which is hereby
incorporated by reference herein.
[0118] For a more detailed description of ribozyme design, see,
Draper, U.S. Ser. No. 08/103,243 filed Aug. 6, 1993, hereby
incorporated by reference herein in its entirety.
[0119] Other embodiments are within the following claims.
Sequence CWU 1
1
21 1 11 RNA Artificial Sequence Description of Artificial Sequence
Synthesized Hammerhead Target. 1 nnnnuhnnnn n 11 2 28 RNA
Artificial Sequence Description of Artificial Sequence Synthesized
Hammerhead Ribozyme. 2 nnnnncugan gagnnnnnnc gaaannnn 28 3 15 RNA
Artificial Sequence Description of Artificial Sequence Synthesized
Hairpin Target. 3 nnnnnnnyng hynnn 15 4 47 RNA Artificial Sequence
Description of Artificial Sequence Synthesized Hairpin Ribozyme. 4
nnnngaagnn nnnnnnnnna aahannnnnn nacauuacnn nnnnnnn 47 5 85 RNA
Artificial Sequence Description of Artificial Sequence Hepatitis
Delta Virus (HDV) Ribozyme. 5 uggccggcau ggucccagcc uccucgcugg
cgccggcugg gcaacauucc gaggggaccg 60 uccccucggu aauggcgaau gggac 85
6 176 RNA Artificial Sequence Description of Artificial Sequence
Neurospora VS RNA Enzyme. 6 gggaaagcuu gcgaagggcg ucgucgcccc
gagcgguagu aagcagggaa cucaccucca 60 auuucaguac ugaaauuguc
guagcaguug acuacuguua ugugauuggu agaggcuaag 120 ugacgguauu
ggcguaaguc aguauugcag cacagcacaa gcccgcuugc gagaau 176 7 13 RNA
Artificial Sequence Description of Artificial Sequence Substrate
for non-nucleotide containing catalytic nucleic acid. 7 gaccgucaga
cgc 13 8 32 RNA Artificial Sequence Description of Artificial
Sequence Non-nucleotide containing catalytic nucleic acid. 8
gcuggucuga ugagguccgg accgaaacgg uc 32 9 15 RNA Artificial Sequence
Description of Artificial Sequence Target for hammerhead ribozyme
targeted against site A (HHA). 9 agggauuaau ggaga 15 10 32 RNA
Artificial Sequence Description of Artificial Sequence Hammerhead
ribozyme targeted against site A (HHA). 10 ucuccaucug augagggaaa
ccgaaaaucc cu 32 11 15 RNA Artificial Sequence Description of
Artificial Sequence Target for hammerhead ribozyme with abasic
substitutions (HHA-a). 11 agggauuaau ggaga 15 12 33 RNA Artificial
Sequence Description of Artificial Sequence Hammerhead ribozyme
with abasic substitutions (HHA-a). 12 ucuccaucng angaggnnnn
ccgaaaaucc cun 33 13 15 RNA Artificial Sequence Description of
Artificial Sequence Target for site B HH ribozyme (HHB). 13
ggagaauugg aaaac 15 14 34 RNA Artificial Sequence Description of
Artificial Sequence Site B HH ribozyme (HHB). 14 guuuucccug
augaggggaa acccgaaauu cucc 34 15 15 RNA Artificial Sequence
Description of Artificial Sequence Target for site B HH ribozyme
with abasic substitutions (HHB-a). 15 ggagaauugg aaaac 15 16 34 RNA
Artificial Sequence Description of Artificial Sequence Site B HH
ribozyme with abasic substitutions (HHB-a). 16 guuuucccng
augaggggaa acccgaaauu cucc 34 17 36 RNA Artificial Sequence
Description of Artificial Sequence Control ribozyme. 17 ucuccaucug
augaggccga aaggccgaaa aucccu 36 18 32 RNA Artificial Sequence
Description of Artificial Sequence Ribozyme A. 18 ucuccaucug
augaggccgg ccgaaaaucc cu 32 19 28 RNA Artificial Sequence
Description of Artificial Sequence Ribozyme C. 19 ucuccaucug
augagcgcga aaaucccu 28 20 36 RNA Artificial Sequence Description of
Artificial Sequence Ribozyme C. 20 ucuccaucug augaggccnn nnggccgaaa
aucccu 36 21 32 RNA Artificial Sequence Description of Artificial
Sequence Ribozyme D. 21 ucuccaucug augagcnnnn gcgaaaaucc cu 32
* * * * *